CN111164485B

CN111164485B - Method and device for optically examining a plurality of microscopic samples

Info

Publication number: CN111164485B
Application number: CN201880063710.5A
Authority: CN
Inventors: T.卡尔克布伦纳; J.西本摩根; T.奥特
Original assignee: Carl Zeiss Microscopy GmbH
Current assignee: Carl Zeiss Microscopy GmbH
Priority date: 2017-09-29
Filing date: 2018-09-25
Publication date: 2023-01-24
Anticipated expiration: 2038-09-25
Also published as: JP2020535421A; US11371927B2; WO2019063539A1; DE102017122718A1; US20200284715A1; CN111164485A; JP7309698B2

Abstract

The invention relates to a method for optically examining a plurality of microscopic samples (1), wherein the samples (1) are transported in a channel from the plurality of samples and are successively guided by means of a fluid in at least one through-flow channel (3), in which the samples advance in a flow direction (23). The sample (1) is illuminated and the light emitted by the sample (1) is detected and analyzed. The invention also relates to a device for carrying out said method. According to the invention, the sample (1) is illuminated in such a way that at least one light sheet (19) having a light sheet plane (22) is aligned with the at least one through-flow channel (3), wherein the light sheet (19) is oriented in such a way that it intersects the at least one through-flow channel (3) in an intersection region (20), and a normal (21) to the light sheet plane (22) and the flow direction (23) enclose an angle different from zero in the intersection region (20). Light emitted by the sample (1) is recorded by imaging detection optics (10), the focal plane of the detection optics (10) being located in the intersection region (20).

Description

Method and device for optically examining a plurality of microscopic samples

Technical Field

The invention relates to a method for optical examination, in particular for operation, for imaging and for analyzing a plurality of microscopic, in particular biological, samples, wherein the samples are transported from a plurality of sample channels and are successively guided by means of a fluid in at least one throughflow channel, and wherein the samples advance in the throughflow channel in a predefined flow direction. The sample is illuminated in the throughflow channel and the light emitted (or reflected) from the sample is detected and analyzed.

The invention also relates to a device for optically inspecting a plurality of microscopic samples, which is particularly suitable for carrying out the aforementioned method. Such a device comprises a channel transport device for channel-transporting the sample and for successively transporting the sample to at least one throughflow channel, in which the sample advances in a flow direction predetermined, for example, by a pumping and discharge device. The apparatus further comprises an illumination device for illuminating the sample in the through-flow channel to emit probe light; the apparatus further comprises detection means for detecting detection light emitted by the sample and analysis means for analyzing said detection light.

Background

Over the past fifty years, flow cytometry has become one of the important measurement methods for examining large populations of cells to obtain quantitative information about cell type and number. The cells are guided essentially individually at a relatively high speed through a light beam for illumination or a device for sample manipulation, for example a voltage. Depending on the cell's properties, such as the cell's structure, shape or staining with e.g. fluorescent markers, the operation leads to different results which are detected and allow conclusions to be drawn about the cell's properties. The term "manipulation" is to be understood here to mean, in addition to mechanical or quasi-mechanical intervention, for example by means of optical tweezers, also a change in the properties of the sample due to external intervention. The light beam may also manipulate the sample if, for example, it is labeled with a fluorescent marker and excited by the light beam to emit fluorescence. In this case, only signals are detected in conventional flow cytometry analysis, but no two-dimensional or three-dimensional image information can be collected, which is desirable for more precise analysis and/or for further diversified classification.

In flow cytometry, a sample is separated from a sample volume, which usually contains a plurality of microscopic samples, which are understood below as individual objects, for example cells, in a carrier liquid, by means of a channel transport device and is usually diverted in microchannels by means of a so-called sheath flow. For example, the separation can be carried out by means of funnel-shaped openings, which can be adjusted in diameter, to the microchannels. Microchannels, which are usually designed at present on microfluidic chips, usually have a diameter which is selected to be so large that cells of typical size between 1 μm and 30 μm are not trapped therein and, on the other hand, so small that all cells passing through the microchannel are also detected by the operating medium, for example a laser beam, without hydrodynamic focusing in the channel at the intersection with the laser beam. If the frequency of the laser beam matches the marker, the sample marked with the fluorescent marker is excited upon passing the laser beam to emit a fluorescent signal, which is recorded by a detector directed to the intersection area between the laser beam and the through-flow channel. In this case, a simple photodetector is generally sufficient, since imaging with an imaging optical system is not necessary for quantitative analysis. For samples not labeled with a fluorescent marker or additionally to fluorescence, the scattered light of the sample in different directions can be recorded.

The detected measured values are evaluated in the analysis unit and information is obtained about the quantification of each analyzed cell. The number of samples analyzable per second is 10 ⁴ Within an order of magnitude, representative information about individual samples, such as cell populations present in multiple samples, can thus be obtained in a minimum amount of time. In this case, a plurality of lasers, which each emit light of a different wavelength, can be positioned at the throughflow channel and can be coupled to a corresponding detector, so that a plurality of different samples can be analyzed. Alternatively to analyzing the fluorescent signal, the scattered light may be analyzed.

Sorting by means of a sorting device is connected to flow cytometry measurement. And sorting the samples into different channels according to the analysis result. If this has not already been done before the analysis, the sample is separated, for example, by means of a shaker arranged in the throughflow direction behind an illumination means, also referred to as excitation means, which separates the liquid flow into small droplets which usually contain only one cell, and then electrostatically sorted, in the case of fluorescence excitation. A summary of flow cytometric analysis methods, including the Sorting mechanism, is disclosed, for example, in the article "Microfluidic Cell Sorting: A Review of the Advances in the Separation of Cells from Debulking to ray Cell Isolation" by C.W. Shields IV et al, lab chip.15 (5), pp 1230-1249, 2015.

Although high sample throughput can be achieved using these methods, imaging is typically not performed because focusing cannot be performed in this speed. One solution is to use an optical train with a fixed focal length and small aperture, which results in an increased depth of field, which also results in a darker image. This disadvantage is at least partially compensated again by special image processing. However, such so-called "Extended-Depth-of-Field" (EDOF) increases the technical effort and thus the costs, and in addition the achievable resolution in the image is small due to the small aperture. Flow cytometers with combined Imaging are for example the imagestream x Mark II Imaging Flow zytomer from Merck KGaA.

Modern flow cytometers generally use the "lab-on-a-chip" method, here microfluidic systems, in which special sample carriers are used which are provided with microstructured channels having an inflow and an outflow. The microstructured channels can be designed differently depending on the application. For example, to generate a reaction, multiple microstructured channels can be crossed, a simple channel being sufficient for simple flow cytometry analysis. Sample carriers with microstructured channels (also referred to as channel carriers) can be arranged on a corresponding sample holder or sample stage of an inverted microscope in order to be able to observe The channels also with The aid of an inverted microscope, as described, for example, in The article "The microscopical cell (MicCell), a top modular flow through System for cell biology, biological research, and nanotechnology" microfluidic Nanofluid 2, p.21-36 2006. This observation is mostly performed confocal and is therefore limited to a small range.

Furthermore, light sheet microscopy methods and devices for examination of biological samples have been established in the prior art during this time. The illumination of the sample is carried out here with a light sheet, i.e. essentially only in one plane, i.e. the plane of the light sheet. The light sheet encloses a largely right-angled angle different from zero with a detection direction which usually coincides with the optical axis of the detection objective. With this technique, also called planar light Microscopy (spimselection Plane Illumination Microscopy), it is possible to produce even thicker spatial recordings of the sample in a comparatively short time. The image-wise, spatially extended representation of the sample can be achieved with light sheet microscopy on the basis of the optical cross-section in combination with a relative movement of the sample in a direction perpendicular to the plane of the cross-section.

SPIM technology is preferably used in Fluorescence Microscopy, also referred to herein as Light Sheet Fluorescence Microscopy (LSFM). The LSFM technique has many advantages over other established methods, such as confocal laser scanning microscopy or two-photon microscopy. Since probing can be done in a wide angle, a larger sample area can be detected faster. Although the resolution is slightly lower than confocal laser scanning microscopy, thicker samples can be analyzed using the LSFM technique for static samples due to the greater penetration depth. Furthermore, the light load of the sample is lowest in this method, which reduces the risk of discoloration and mainly reduces the photo damage of the sample, since the sample is only irradiated by a thin light sheet at a non-zero angle with respect to the probing direction.

The light sheet is generated both statically, for example by means of a cylindrical lens, and quasi-statically by means of a light beam which rapidly scans the sample in a plane. The light sheet illumination is produced in that the light beam is subjected to a very rapid relative movement with respect to the sample to be observed, and is thereby aligned with one another a plurality of times one after the other in time. Various methods are used for forming the light sheet, so-called Sinc may be used ³ Light sheets, produced on the basis of Mathieu rays or Bessel rays, or even by means of a grid, as described for example in US 2013/286181 A1 in the literature.

When a quasi-static light sheet is used, the integration time of the connected camera (on the sensor of which the sample is finally imaged) is selected such that the scanning ends within the integration time. Instead of a camera with a two-dimensional sensor, it is also possible to use a line sensor in combination with rescanning in the detection optics. SPIM technology is now described repeatedly in the literature, for example in DE 102 57 423 A1 and WO 102/053558 A1 on the basis thereof, or in the review article "Selective Plane Illumination Microscopy Techniques in Development Biology", huisken et al, 2009, journal Development Bd136, 1963 and beyond. In documents WO 2012/1 10488 A2 and WO 2012/122027 A2, a structure is described in which the illumination objective and the detection objective are perpendicular to one another and are each directed at the sample from above at an angle of 45 °. The sample is for example located in a petri dish. Devices for inverting light sheet microscopy are also known in the prior art, for example from the documents DE 10 2013 107 A1, DE 10 2013 112 A1 or DE 10 2013 112 600 A1. Since in light sheet microscopes with an inverted layout the light passes obliquely through the sample carrier, it is necessary to use a correction element in order to compensate for the aberrations caused by the oblique light passing by means of the correction element, as described in the above-mentioned document.

Disclosure of Invention

The object of the present invention is to improve a method and a device of the type mentioned at the outset such that, on the one hand, the sensitivity and flexibility are higher compared to flow cytometry methods and devices known from the prior art, and, on the other hand, image information is provided in the greatest possible detail.

In a method of the type mentioned at the outset, the above-mentioned object is achieved in that the sample is illuminated in such a way that at least one light sheet with a light sheet plane is directed at least one through-flow channel and is oriented in such a way that the light sheet intersects the at least one through-flow channel in an intersection region and a normal to the light sheet plane encloses an angle different from zero with the flow direction in the intersection region. The light emitted by the sample is recorded or detected by imaging (i.e. image-wise and planar detection) detection optics, the focal plane of which lies in the intersection region.

The emitted light is, for example, scattered light or reflected light, in which case the sample passively emits light without being excited for this purpose. It is particularly preferred that the method can also be used on samples which are labeled with fluorescent markers before examination, which makes identification of e.g. cells easier. These samples are excited by the at least one light sheet to emit a fluorescent signal, which is recorded by the detection optics. In this case, the sample actively emits light.

In this case, the sample with the fluorescent marker is not irradiated or excited by a simple laser beam, but by a light sheet, which is also generated by a laser and is oriented such that it intersects at least one through-flow channel in the intersection region. The term "intersection region" is to be understood here as: in an ideal light sheet with an infinitely small thickness in the direction of the normal to the light sheet plane, the intersection area is a two-dimensional intersection surface in the light sheet plane, which results from the intersection of the volume of the through-flow channel with the light sheet, which is delimited by the channel edges. In reality, however, the light sheet has a finite dimension along the normal to the plane of the light sheet, with an upper limit on the thickness of the light sheet between 5 μm and 30 μm, which, although significantly smaller than the dimension in the plane of the light sheet, achieves that the intersection region is an intersection volume whose thickness along the normal to the plane of the light sheet is about 0.4 μm to 3 μm in the best focused region, inclusive. An important advantage of the use of an optical sheet in the case of corresponding focusing is its planar dimensions, which, when using, for example, a channel carrier with a plurality of flow-through channels arranged in parallel, makes it possible for even a plurality of channels to be analyzed simultaneously,

in principle, if only quantitative analysis is required, the light sheet can also be oriented perpendicularly to the flow direction for illumination and in particular for generating a fluorescence signal, which can be detected and analyzed in the usual manner, for example by means of photodetectors, i.e. non-imaging detection optics, wherein advantageously a plurality of throughflow channels can be analyzed simultaneously compared to the use of a single laser beam. The SPIM device thus takes over only the light sheet, while the imaging detection optics, i.e. the image generation, are omitted.

A particularly advantageous aspect of the invention is, however, the oblique orientation of the light sheet, meaning that the normal to the plane of the light sheet encloses an angle with the flow direction which is not zero. In this case, in the case of a generally substantially tubular through-flow channel, the flow direction is oriented in the longitudinal dimension of the channel, i.e. perpendicular to its cross section and, depending on the direction in which the through-flow channel is through-flowing. The flow direction and in particular the value of the flow speed can be controlled from the outside, for example, with a corresponding pump device port.

What is achieved by an angle other than zero is that the detection optics, as they are usually used in slide microscopy, are fitted and positioned in such a way that the focal plane (or focal plane) lies in the intersection region, ideally directly in the plane of the slide or at least parallel thereto, so that the entire inclined cross section of the through-flow channel in the intersection region lies in the focal plane. Since the entire cross section of the throughflow channel is detected, larger channels can be used, i.e. the channel size can be selected independently of the sample size. In this way, it is possible to record, in a snapshot, an intersection image of the sample in the intersection region, i.e. a two-dimensional image of the sample passing through the intersection region, which is typical for light sheet microscopy. In addition to a large or even entirely inclined cross section covering the through-flow channel, the inclined orientation of the light sheet with respect to the flow direction advantageously allows the use of objective lenses with a large numerical aperture and a small working distance in microfluidics. The detection objective can be used not only for generating images, but also for recording fluorescence signals for quantitative analysis, as it is done in flow cytometry experiments, without the need for additional detection means. The imaging detection objective therefore not only records the fluorescence signals, but at the same time also images the sample passing through the intersection region in the focal plane onto the respective area sensor at least in a temporally recorded manner, so that at least one two-dimensional recording of the sample can be produced when passing through the intersection region.

Due to the oblique orientation of the light sheet with respect to the flow direction, sectional images through the entire sample are obtained even for instantaneous shots. However, in conjunction with the flow speed and the flow direction, an image stack consisting of individual images of the sample flowing through the intersection region can be recorded temporally continuously, and the individual images of the image stack can optionally constitute a view in space.

Furthermore, the detection optics are always focused on the plane of the light sheet or on a plane in the intersection region without further measures, so that additional measures, for example for increasing the depth of field (EDOF), can be dispensed with. In flow cytometry using a laser beam, the laser beam usually covers only a partial region of the through-flow channel and particles must be focused on this partial region by means of a corresponding flow focusing, whereas the light sheet usually covers the entire through-flow channel, or at least a large part thereof, in the cross section or intersection region, so that particle focusing can be dispensed with here. In principle, due to the imaging, it is also possible to operate and analyze a plurality of samples, in particular cells, which are located simultaneously in the intersection region in the snapshot, in the flow channel having a larger cross section. In contrast to conventional flow cytometry, the exciting laser beam does not have to be focused in order to generate a maximum signal, which is necessary there to focus the particles, since the diameter of the throughflow channel cannot be reduced to the diameter of the focused laser beam.

In the prior art, imaging in flow cytometry can only be achieved by the extended depth of field method (EDOF method), which requires the use of a small aperture for imaging, while these measures can be dispensed with when using light sheet microscopy devices for illumination and detection: the detection objective is focused onto the plane of the light sheet with a depth of field corresponding to the thickness of the intersection in the direction normal to the plane of the light sheet and substantially smaller than the dimension of the sample. It is therefore possible to use an objective with a large aperture angle (or viewing angle) in the detection optics, which achieves a high resolving power, which substantially increases with the aperture angle of the objective. By using an immersion medium the numerical aperture of the objective lens can be further increased and the resolution can be further improved.

The method can even be combined with a masked or off-axis illumination (oblique illumination) method to increase contrast. The off-axis illumination is coupled into the illumination objective, for example, in a further color, alternatively a separate objective can also be used. In this way, a comparative recording of the sample can be established, for which spectrally separated channels are used for illumination and detection. The image data may be combined into an overall image of the sample with higher contrast.

After analyzing the fluorescent signal from these signals, the sample can be sorted using measures known in the art.

Since the light sheet can cover a significantly larger area than the diameter of the through-flow channels for the microfluid, the light sheet can be directly oriented such that it intersects a plurality of through-flow channels. This allows a significant increase in throughput and an increase in efficiency of the method for flow cytometry and image analysis.

Furthermore, it is also possible to illuminate the sample with a plurality of light sheets, wherein the planes of the plurality of light sheets are preferably parallel to each other. In this case, for example, a plurality of identical light segments can be arranged one after the other in the flow direction, which enables repeated observation of the same object, i.e. of a sample separated from the total amount of sample. It is also possible to use a plurality of light sheets with different spectral characteristics for, for example, observing polychromatic excitation. The sheets can also be superimposed on each other, with simultaneous excitation of the multiple colors. The optical operation can also be observed in a time-course manner if identical or different light sheets are arranged one after the other. For example, a first light sheet, which causes a reaction in the sample. The second light sheet is used for observing the first property, if necessary further light sheets can be used for observing further properties, which can even be superimposed. By setting the flow rate, e.g. via a valve, the time required for the sample to be displaced between the first and second light sheet can be set.

Depending on the properties to be examined, the second light sheet or, if appropriate, further light sheets can be superimposed on one another or arranged spaced apart from one another in the flow direction. There is a spatial distance between the first and second light sheets. Furthermore, a corresponding design of the at least one through-flow channel makes it possible to make the light sheet intersect the at least one through-flow channel several times in the intersection region, so that the temporal behavior of the sample can be checked. This can be achieved, for example, by zigzag-shaped through-flow channels, even curved arrangements of the through-flow channels being conceivable.

Furthermore, when using a plurality of through-flow channels, all parallel channels or groups of channels can be assigned individualized spectral characteristics by using special filter devices or process emission filters.

The object is achieved with a device of the type mentioned at the outset in that the illumination means comprise illumination optics for generating at least one light sheet with a light sheet plane, wherein the light sheet is oriented relative to the flow-through channel in such a way that the light sheet intersects the flow-through channel in an intersection region and a normal to the light sheet plane encloses an angle different from zero with the flow direction in the intersection region, in order to illuminate the sample in the intersection region. The detection device comprises imaging detection optics, the focal plane of which is located in the intersection region.

The description based on the device features applies analogously to the corresponding methods in respect of these features, while the method features correspondingly represent functional features of the described devices.

Suitably, the illumination device is designed to emit light of a specific wavelength or wavelength range for exciting a sample labeled with a fluorescent marker, which sample then emits a fluorescent signal. The fluorescence signal is then detected planarly by the imaging detection optics. The detector means may however also comprise further detectors, for example pure photodetectors, which are used to detect fluorescent signals and/or scattered light if quantitative analysis is the focus.

Advantageously, in the device according to the invention, the at least one through-flow channel is designed on a replaceable channel carrier, for example a microfluidic chip. This enables targeted adjustment of different samples and analysis methods. For example, a plurality of parallel through-flow channels can be arranged on the channel carrier, which intersect the light sheet, i.e. all through-flow channels have an intersection area with the light sheet. This enables a greater sample throughput due to the parallelization and can be used in particular when a plurality of samples are to be analyzed and the emphasis is not to be represented imagewise, which can likewise be integrated directly with the area detector: using the detection optics and the camera connected thereto, the defined regions of the detector can be assigned in each case to a defined channel on the area detector of the camera, so that all throughflow channels can be evaluated separately. If only quantitative analysis is performed, all the throughflow channels on the channel carrier can have a common feed line and a common discharge line. However, it is also possible to provide all channels with their own input and/or output lines, or to combine the channels in groups, depending on the analysis to be carried out.

In the at least one throughflow channel, a sorting unit can be arranged downstream of the light sheet in the flow direction, with which the sample is guided, depending on the result of the analysis, onto one of the plurality of branch channels adjoining the throughflow channel. The sorting can be performed using methods known in the art, for example, optically, acoustically or electrophoretically.

In order to observe the temporal behavior of the marked or manipulated sample, multiple observations of the sample are required. In a preferred embodiment of the invention, the illumination device therefore comprises an illumination optics or a plurality of illumination optics for producing a plurality of light sheets. The light sheets are parallel to one another with their light sheet planes. The illumination optics produce light sheets of different colors, respectively, or light sheets of the same color, according to the kind of experiment. The light sheets of several colors can even be designed superimposed to produce multiple excitations in at least one flow channel. In other cases, operations, such as the generation of reactions or the excitation of fluorescence, are carried out in the region of the intersection of the first light sheet with the at least one through-flow channel, which is observed with the second light sheet. In a particularly preferred embodiment, a plurality of light sheets are produced by the same illumination optics. For the two light sheets, this can be done, for example, by means of a controlled scanner, which switches between two parallel light sheet planes. The scanner is coupled via a controller to a switching device, which ensures that the light sheet is switched off during the switching process.

Alternatively or additionally, it is possible to design the flow-through channels on the channel carrier such that the flow-through channels form a plurality of intersection regions with the light sheet, which can be done, for example, by designing at least one flow-through channel in a zigzag manner. If the spectral range of the light sheets is equally suitable for excitation and detection, only one light sheet is required in this case for observing the temporal sequence of the reactions, in other cases a plurality of light sheets of different colors being superimposed.

In other embodiments, the throughflow channel is designed such that its diameter varies in the direction of flow, which enables mechanical manipulation of the sample in the sense of deformation. The primary sample is then observed, for example, before the deformation and during the deformed state, for which purpose two light sheets are used if the flow direction is not changed, or a single light sheet is used if at least one through-flow channel is designed such that the flow direction is changed relative to a superordinate coordinate system, as is the case in the case of a meander-shaped through-flow channel.

It is advantageous that the value of the flow speed in the at least one throughflow channel can even be set variably by means of a speed regulation device and adapted to the experimental conditions. The speed can be adjusted, for example, by valves on the feed line and/or the discharge line.

Furthermore, if an image stack is generated from at least three images at different positions inside the object, the phase information with respect to the detected light can be determined by means of a so-called phase recovery algorithm, for example the intensity propagation equation (TIE). Since the sample, object, flows through the focal plane of the detection optics, only at least 3 images need to be recorded at suitable time intervals, unlike in conventional microscopy, where the sample or objective lens must be moved along the optical axis. Furthermore, the apparatus may be combined with additional detection apparatus and cameras, such as a normal reflection light microscope or other light sheet microscope device.

The features mentioned above and those yet to be explained below can, of course, be used not only in the combination stated but also in other combinations or alone without departing from the scope of the invention.

Drawings

The invention is further elucidated below, for example, on the basis of the appended drawings, which disclose essential features of the invention. Wherein:

figure 1 shows a schematic structure of an apparatus for optically processing a plurality of samples,

figure 2 shows details of the channel carrier and the light sheet position,

figure 3 shows a second design of such a device,

figure 4 shows a third design of such a device,

figure 5 shows the design of the channel carrier,

figure 6 shows a second design of the channel carrier,

figure 7 shows a third design of the channel carrier,

fig. 8a, b show two further embodiments of the channel carrier.

Detailed Description

Fig. 1 first shows the basic configuration of an apparatus for optically manipulating a plurality of biological samples 1 labeled with fluorescent markers. A sample 1, for example a plurality of different cells originating from a tissue sample, usually present in a carrier liquid in a sample total amount, is first prepared, i.e. labelled with a fluorescent label, and then transported into a channel transport device 2, wherein the sample 1 is transported by a channel and transported to at least one through-flow channel 3. The channel transport device 2, which is also referred to as a separating device, can have, for example, a funnel-shaped opening in the direction of the throughflow channel 3, the size of which is such that the sample can only pass through it individually. This can be assisted by means of fluidic measures, wherein advantageously the diameter of the opening can even be varied. Typical samples are cells which usually have a size of 1 to 30 μm, the diameter of at least one through-flow channel 3 of usually tubular configuration matching these cell sizes and usually being only slightly larger than the largest diameter of the sample 1 to be examined, so that they do not block the through-flow channel 3. In this case, if it is ensured by corresponding measures that the samples 1 enter the at least one through-flow channel 3 as far as possible one after the other, a through-flow channel 3 with a larger diameter (for example 40 μm and larger) can also be used for smaller samples with a diameter of less than 5 μm, which can be achieved, for example, by the variable size of the inlet opening at the transition from the channel transport device 2 to the through-flow channel 3. However, due to the special illumination and detection (described in more detail below), it is also possible for a plurality of samples 1 to be located in the through-flow channel 3 simultaneously or side by side. Furthermore, the at least one throughflow channel 3 is coupled to a pump device, not shown here, which is used to generate a flow, here indicated by the flow direction 23. By means of corresponding control and by means of valves, the flow speed and/or even the flow direction can be changed depending on the analysis to be carried out.

At least one flow-through channel 3 is formed on or in a channel carrier 4, wherein it can be a microfluidic chip, for example. These microfluidic chips can be specifically configured or produced for a plurality of analyses and/or experiments, and the channel carrier 4 is therefore preferably replaceable. The through-flow channel 3 is connected to the channel transport device 2 by means of a hose 5, on the other end of the through-flow channel 3 in the channel carrier 4 there also being mounted a hose 5 for discharging the samples 1, which can be sorted, transported to further analysis or experiments, or alternatively disposed of, if necessary.

The channel carrier 4 can be moved in all three spatial directions, for which purpose it is arranged here on a correspondingly moving sample table 6, which is additionally rotatable in a plane defined by a bearing surface 7 of the sample table 6. The flow direction 23 in the at least one through-flow channel 3 also lies in this plane at least in the region in which the analysis takes place.

In the at least one through-flow channel 3, the sample 1 flows in the flow direction 23 between the supply line and the discharge line at an average vector flow velocity, wherein it is not excluded that the sample 1 is stopped in place during a short period of time during the manipulation of the velocity. When the through-flow channel 3 has a tubular cross section, the flow direction 23 is in each case along the channel, i.e. parallel to the longitudinal axis of the tubular channel.

For carrying out the analysis, the apparatus has an illumination device with which the sample 1 is illuminated and which is preferably also designed here as an excitation device for exciting the sample 1 in the at least one through-flow channel 3 to emit a fluorescence signal. The sample emits probe light, which can be done passively in the form of scattered light, but can also be done actively in the form of emitted fluorescent signals. The detection light is detected with a detection means which is also part of the apparatus. The device furthermore has an evaluation means for evaluating the detected detection light, for example the detected fluorescence signal. In this case, for example, a correspondingly programmed computer can be provided as an evaluation unit with an output unit, for example a screen.

In the apparatus shown in fig. 1, the illumination means or excitation means and detection means are realized by a light sheet microscope 8, which is shown here in an inverted configuration. This inverted configuration is advantageous if the construction above the sample stage 6 requires too much space, for example due to the hose 5 and the instruments connecting the hoses, it being possible, but not mandatory, to envisage the light sheet microscope being arranged above the channel carrier 4. The light sheet microscope 8 shown here comprises illumination optics 9 and detection optics 10. The illumination optics 9 are shown on the left side of fig. 1. The light originating from the laser module 11 (in this case, for example, a plurality of lasers of different wavelengths are provided or can be selected between different wavelengths, wherein even a plurality of wavelengths can be selected simultaneously) is deflected by the light shaping module 12 and the scanning module 13 (for example, for generating a quasi-static light sheet and/or for angle setting) onto the illumination objective 14, and the illumination objective 14 images the light sheet into the light sheet plane into the channel carrier 4 and the through-flow channel 3, which in this case coincides with the optical axis of the illumination objective 14. The focal point of the illumination objective 14, i.e. the position of the light sheet with the narrowest extent, can be controlled by means of a drive, for example by means of a piezoelectric drive 15, such that the focal point is located in the through-flow channel, for example, if only one through-flow channel is used. Alternatively or additionally, the sample table 6 can be adjusted.

On the right, an exemplary detection optics 10 is shown, which comprises a detection objective 16, the detection objective 16 being adjusted similarly to the illumination objective 14 by means of a drive, here likewise by means of a piezo drive 17. The optical axis of the detection objective 16 encloses an angle with the light sheet plane, which is not zero, here a right angle, in which the optical axis of the illumination objective 14 lies. However, this is not absolutely necessary: in order to operate the method, an angle between the plane of the light sheet and the optical axis of the detection objective 16 that is not zero is sufficient. However, the advantage of arranging the two optical axes of the objective at an angle of 90 ° with respect to one another is that the focal plane of the detection objective 16 is then identical to the light sheet plane, so that the entire area of the light sheet plane can be clearly imaged if detected by the detection objective 16, i.e. in this case through the entire, oblique cross section of the through-flow channel 3. The detection light emitted from the sample 1, for example the fluorescence light emitted from the sample after corresponding excitation, is diverted by the detection objective 16 onto the detection 18, where a plurality of detection modules can be used with the aid of a beam splitter. When the sample is illuminated simultaneously or quasi-simultaneously with a light sheet comprising a plurality of wavelengths, the detection can then be carried out differently, for example, depending on the wavelength. The flat detectors, which usually are located in the detection module 18, record the intensity and, if necessary, the color and convert them into corresponding electrical signals, which are then transmitted to the evaluation unit and the image processing unit and processed there.

Fig. 2 shows the channel carrier 4 in detail. At least one flow channel 3 is designed in a channel carrier 4, for example a microfluidic chip, which is here usually a transparent, plate-shaped body with two large surface sections parallel to one another and an edge surface section, which connects the two large surface sections. The at least one through-flow channel 3 is designed as a cavity which is open to both sides in the transparent plate-shaped body. In the device shown, the light sheet 19 is oriented such that it intersects the at least one flow-through channel 3 in an intersection region 20, in which the sample 1 is excited to emit a fluorescence signal, for example, and a normal 21 to a light sheet plane 22 encloses an angle different from zero with the flow direction 23 in the intersection region 20. The transparent material of the channel carrier 4 is located between the intersection region 20 and the illumination objective 14 or the detection objective 16, and, due to the selected arrangement, the optical axes of the detection objective 16 and the illumination objective 14 are arranged not at right angles to the interface between the channel carrier 4 and the throughflow channel 3 and the interface between the bottom side of the channel carrier 4 and the medium located therebelow, but at an inclination. This results in aberrations. To correct these aberrations, the light sheet microscope 8 can have different correction devices, which are shown by way of example in fig. 1, and which can be used individually or in combination. For example, a special lens, a so-called "virtual relay, virtuelle Relays", may be attached in front of the objective lens. Here, it is a free anamorphic lens which is designed to correct aberrations with a predefined thickness of the channel carrier 4 (here between the throughflow channel 3 and the bottom, i.e. bottom side, of the channel carrier 4) and with a predefined thickness of a medium (e.g. air or immersion medium) located between the free anamorphic lens and the channel carrier. Aberrations caused by the immersion medium can be corrected by means of the meniscus lens, wherein corrections for a plurality of immersion media can also be carried out here depending on the design using the meniscus lens. However, aberrations caused by the passage of light through the channel carrier 4 between the bottom side of the channel carrier 4 and the through-flow channels 3 which are skew-based cannot be corrected with a meniscus lens. Finally, a wavefront manipulator, a so-called Alvarez plate, can be used for correcting both aberrations. The range of thicknesses of the virtual repeater can be extended, for example, by means of a wavefront manipulator.

In the embodiment shown here, on the one hand, a settable wavefront manipulator 24 is arranged in each case, for example, in the illumination objective 14 and in the detection objective 16. On the other hand, the virtual relay 25 is located between the objective of the light sheet microscope 8 and the channel carrier 4, the virtual relay 25 being shown here as one lens, but may also comprise a plurality of lenses. For example air 26 or immersion liquid 27, is located as a medium between the virtual repeater 25 and the bottom side of the tunnel carrier 4. The correction means (virtual relay 25 or meniscus lens 28 and wavefront manipulator 24) can be used jointly or individually, if necessary depending on the medium on the bottom side of the channel carrier 4. Furthermore, it is of course also possible to use further correction mechanisms, for example further front lenses or free anamorphic lenses, which are integrated in the objective.

The sample 1 advances in the flow direction 23 in the at least one through-flow channel 3 as shown in fig. 2, and if the sample 1 is appropriately marked beforehand, fluorescence is excited by the light sheet 19 in the intersection region 20. The intersection region 19 is a smaller volume, since the light sheet also has a limited dimension perpendicular to the plane of the light sheet in the region of maximum focus, which is however significantly smaller than the dimension of the light sheet in the plane, relative to which it can be ignored. However, for the sake of clarity, the intersection region is shown here slightly enlarged. The fluorescence signal is detected by the detection optics 10 and quantitatively evaluated by an analysis means not shown, as is common in the art. In addition to the fluorescence signal, however, it is also possible to record or record a complete image of the sample 1 and, in addition, to record the image stack without mechanically moving parts, since the sample 1 passes through the focal plane of the detection objective 16, which generally coincides with the light sheet plane 22, in the flow direction 23, depending on the flow speed. The recorded image data, for example, the shape, morphology, spatial structure, which can additionally be used for further analysis and, if necessary, even for a corresponding sorting of the samples according to further parameters, in contrast to fluorescence, are present here at a greater resolution than is achievable in the prior art. No separate focusing of the sample is required. Even the sample does not have to be focused in the flow channel 3 in terms of flow technology, since the light sheet 19 covers the flow channel 3 in its cross section. This has the advantage that even larger through-flow channels 3 can be used and that a larger cell density can be measured, especially when spatial imaging is required.

A combination of the device with further detection means is possible, examples of which are shown in fig. 3 and 4. In addition to the light sheet microscope 8 shown here in a simplified manner, fig. 3 shows a conventionally arranged microscope objective 29, which is connected to a camera 30. It is oriented relative to the flow-through channel 3 or the transparent, plate-shaped channel carrier 4 in such a way that it is oriented perpendicular to the interface, so that no correction of aberrations arising from the inclination of the incident light is required here. With the aid of elements (EDOF elements) 31 which can be introduced into the beam path and can also be removed therefrom for increasing the depth of field, two-dimensional images with lower resolution can also be generated in this way. In addition to the inverted light sheet microscope 8, fig. 4 shows a further light sheet microscope 32 arranged above the flow-through channel 3, which light sheets intersect. The same configuration can be used for the two

light sheet microscopes

8, 32, but it is entirely feasible to use different configurations. The difference in this case is the correction means and the medium between the objective and the throughflow channel 3. The light sheets 19 may have different colors, which may be produced in different ways. For example, it is possible to use static light sheets produced by means of cylindrical lenses or light sheets based on other forms, so-called Sinc3 light sheets, light sheets produced by means of Mathieu rays or Bessel rays, or even grid light sheets produced by means of grids.

The following describes different channel carriers or microfluidic chips which are particularly suitable for use with planar extended light sheet illumination. Such a channel carrier is shown in fig. 5-8.

Since the light sheet has a dimension in the plane which is larger than the diameter of the through-flow channels 3 (for example 400 μm and larger widths and lengths in the range from 20 μm to 200 μm depending on the chosen illumination optics) and since the focal plane of the detection objective 16 lies in the light sheet plane 22, a channel carrier 4 can be used on which a plurality of parallel through-flow channels 3 are arranged. An example of this is shown in figure 5. In this case, the throughflow channel 3 can comprise a common feed line and discharge line if the samples 1 are derived from a single group of samples. It is also possible, however, to combine the throughflow channels 3 into a plurality of groups, wherein the groups themselves have identical supply and discharge lines, but each group has its own supply and discharge line. This enables parallel examination of samples 1 originating from different total amounts of sample. Furthermore, a separate supply line and a separate discharge line can be assigned to all flow channels 3. Even the discharge lines for all through-flow channels 3 can be common even when they are grouped differently, for example for the case in which the samples 1 are then no longer used but are to be conveyed to a common processing container.

The light sheet 19 may be a multicolored light sheet, which is particularly advantageous if samples 1 originating from different differently marked sample quantities are to be examined in parallel, or if individual samples 1, for example cells, are marked with a plurality of pigments, which fluoresce in different wavelengths depending on the excitation wavelength; light sheets of the same or different colors can also be arranged parallel to one another at a predetermined mutual distance, which can be detected in a corresponding design with a common detection optics.

The result of the analysis of the data detected by the detection optics 10 can be used to sort the sample 1 subsequently by means of the sorting unit after it has passed through the light sheet. An example for such a channel carrier 4 is shown in fig. 6. The sorting unit includes a sorting mechanism 33 and a branch channel 34. The sorting mechanism 33 can be designed in a manner known in the art, which can, for example, manipulate the sample 1 electrophoretically, acoustically or optically, or deflect the sample from its flow direction so that it is diverted into one of the plurality of branch channels 34. This can even be done for multiple channels simultaneously, since the light sheet has a larger area.

The fact that the optical slide has a larger areal dimension or extension compared to the channel cross section can be used to check the temporal behavior of the sample wafer 1 if, for example, a channel carrier 4 is used which has a meandering flow channel 3 which intersects the optical slide 19 a number of times, so that a number of intersection regions 20 which are arranged one behind the other in the flow direction 23 are produced in the flow channel 3. This is shown for example in fig. 7. The intersection area 20 is shaded. This design of the channel carrier 4 in combination with the above-described apparatus is particularly suitable for investigating the temporal course of the behavior of the sample after manipulation. For this purpose, it is advantageous to superimpose light segments of different colors and, if necessary, to control them over time, if necessary in an experimental or analytical manner. In the configuration shown in fig. 7, the sample 1 first enters the through-flow channel 3 from the left and passes the first of the intersection areas 20. Where an operation is performed, which may for example simply be the excitation of fluorescence emissions. Other possible methods of operation are, for example, photoactivation (i.e. switching sample 1 to a state capable of fluorescence) and photorelease (unincaging), photoconversion (switching sample 1 from a state emitting a fluorescence signal at a first wavelength to a state emitting a fluorescence signal at a second wavelength), or FRAP (fluorescence bleach recovery technique). Here, a local high-intensity illumination is applied inside the sample 1, for example inside a cell, which locally triggers a photobleaching process with respect to fluorescence at this location. Shortly thereafter, the excitation is weakly induced at the other locations and an image is recorded which is evaluated to what extent the fluorescence is built up again by local diffusion. If necessary, additional light sheets can be used to trigger additional reactions. The sample 1 leaves this intersection region 20 again and is guided by the flow further in the flow-through channel 3 until it reaches the next intersection region 20. There, a primary observation or a further operation can be carried out with the aid of the light sheet or of another light sheet. In the further intersection region 20, further observations can be carried out with further light sheets if necessary. Basically, it is possible for all superimposed color light sheets to be permanently connected, so that the temporal control of the light sheets between each two intersection regions 20 can be carried out depending on the flow rate and the length of the section of the flow channel 3, so that only the light sheets required there are switched on at the moment the sample 1 passes through the intersection regions 20. This method is used in particular when studying relatively small samples which flow through the through-flow channel 3 at a not too high velocity.

Two further embodiments for the channel carrier 4 are shown in fig. 8a and 8 b. In this case, the at least one through-flow channel 3 changes its diameter in the flow direction 23, narrowing it in this case. This narrowing can be used to mechanically deform the sample 1, in particular the cells. By corresponding positioning of the light sheet (as shown, for example, in fig. 8 a), an image of the sample before the deformation and an image during the deformation can be recorded, wherein a spatial image of the deformed cells can be generated directly by the flow. Alternatively, the flow channel 3 is diverted after the constriction and is returned, so that the deformed sample passes through the same light sheet again. The through-flow channel 3 widens again and further images can be recorded, by means of which it can be analyzed whether permanent deformations have occurred.

The device can be used for optically manipulating and analyzing biological samples, in particular for a plurality of samples, which are labeled with fluorescent markers in a short time. In addition to the quantitative analysis, it is possible to generate the image data in high resolution not only in two dimensions but also in a spatial representation, so that even high-resolution images for detailed analysis are available. In this way, it is possible to generate further parameters for possible sorting of the samples. A high sample throughput is achieved here not only for the analysis but also for the detailed analysis of all individual samples.

List of reference numerals

1 sample of

2-channel transmission device

3 through-flow channel

4-channel carrier

5 Flexible pipe

6 sample stage

7 bearing surface

8 light sheet microscope

9 illumination optics

10 detection optics

11 laser module

12-ray shaping module

13 scanning module

14 illumination objective

15 piezoelectric driving device

16 detection objective

17 piezoelectric driving device

18 detection module

19 light sheet

20 intersection region

Normal of 21 light sheet plane

22 light sheet

23 direction of flow

24 wave front manipulator

25 virtual repeater

26 air (air)

27 immersion liquid

28 meniscus lens

29 microscope objective

30 vidicon

31EDOF element

32 additional light sheet microscope

33 sorting mechanism

34 branching off the channel.

Claims

1. A method for optically examining a plurality of microscopic samples (1),

the samples (1) are conveyed in a channel from a plurality of samples and are successively guided by means of a fluid in at least one through-flow channel (3) in which the samples advance in a predefined flow direction (23),

-illuminating the sample (1),

-detecting and analyzing light emanating from the sample (1), characterized in that,

-illuminating the sample (1) in such a way that at least one light sheet (19) with a light sheet plane (22) is aligned with the at least one through-flow channel (3), and the light sheet (19) is oriented in such a way that the light sheet intersects the at least one through-flow channel (3) in an intersection region (20), and a normal (21) to the light sheet plane (22) and a flow direction (23) enclose an angle different from zero in the intersection region (20), and

-the light emitted by the sample (1) is recorded by imaging detection optics (10) whose focal plane is located in the intersection region (20) and in the light sheet plane (22) or parallel to the light sheet plane (22) such that the cross section of the at least one through-flow channel (3) is located completely in the intersection region (20) and in the focal plane,

wherein, for checking the temporal behavior of the sample, the at least one light sheet (19) intersects the at least one through-flow channel (3) a plurality of times in a plurality of intersection regions (20), or the at least one light sheet (19) intersects the plurality of through-flow channels (3).

2. The method according to claim 1, characterized in that the detection optics (10) detect images of the individual samples (1) flowing through the intersection region (20) or detect image stacks consisting of individual images of the individual samples (1) flowing through the intersection region (20) consecutively in time.

3. The method of claim 2, wherein the individual images of the image stack constitute a display in space.

4. Method according to one of claims 1 to 3, characterized in that the sample (1) is labeled with a fluorescent marker before the examination, the sample is excited by the at least one light sheet (19) to emit a fluorescent signal, and the fluorescent signal is detected and analyzed.

5. A method according to one of claims 1 to 3, characterized in that sorting of said sample (1) is performed on the basis of said analysis of the light detected by the detection optics or on the basis of information of the image.

6. Method according to claim 5, characterized in that the sorting of the samples (1) is performed on the basis of information of the images and/or fluorescence signals.

7. A method according to one of claims 1 to 3, characterized in that the sample (1) is illuminated with a plurality of light sheets (19).

8. The method according to claim 7, characterized in that the sample (1) is illuminated with a plurality of light sheets (19) of different wavelengths.

9. An apparatus for optical examination of a plurality of microscopic samples (1), comprising

-a channel transport device (2) for transporting samples in channels and for successively transporting samples (1) to at least one through-flow channel (3) in which the samples (1) advance in a predefined flow direction,

-illumination means for illuminating the sample (1) in the at least one through-flow channel (3) to emit probe light,

-detection means for detecting detection light emitted by the sample (1),

-analyzing means for analyzing the probe light, characterized in that,

-the illumination device comprises an illumination optics (9) for generating at least one light sheet (19) with a light sheet plane (22), wherein the light sheet (19) is oriented with respect to the through-flow channel (3) such that the light sheet intersects the through-flow channel (3) in an intersection region (20) and a normal (21) to the light sheet plane (22) encloses an angle with a flow direction (23) in the intersection region (20) that is not zero, thereby illuminating the sample (1) in the intersection region (20), and

the detection device comprises an imaging detection optics (10), the focal plane of which is located in the intersection region (20) and in the light sheet plane (22) or parallel to the light sheet plane (22) such that the cross section of the at least one through-flow channel (3) is located completely in the intersection region (20) and in the focal plane, wherein the at least one through-flow channel (3) is designed in a meandering manner with respect to the position of the light sheet (19) in order to form a plurality of intersection regions (20) with the light sheet.

10. Device according to claim 9, characterized in that the at least one through-flow channel (3) is designed on a replaceable channel carrier (4).

11. Device according to claim 10, characterized in that a plurality of parallel through-flow channels (3) are arranged on the channel carrier (4).

12. Device according to claim 11, characterized in that the parallel throughflow channels (3) have mutually separate feed lines and/or discharge lines and respectively have an intersection region (20) with the light sheets (19).

13. Device according to claim 9 or 10, characterized in that a sorting unit is arranged behind the light sheet (19) in the flow direction (23) and/or an operating unit is arranged in front of or behind the light sheet (19).

14. Device according to claim 9 or 10, characterized in that the illumination means are designed to emit light for exciting the sample (1) labeled with a fluorescent marker to emit a fluorescent signal.

15. The apparatus according to claim 14, wherein the detection means comprises a further detector for detecting fluorescent signals and/or scattered light.

16. An apparatus according to claim 9 or 10, wherein the detection means comprises a further detector for detecting fluorescent signals and/or scattered light.

17. The device according to claim 9 or 10, characterized in that the illumination means comprise one illumination optics (9) or a plurality of illumination optics (9) for generating a plurality of light sheets (19), the light sheet planes of which are parallel to each other.

18. The apparatus of claim 17, wherein all of the light sheets have different colors.

19. Device according to claim 9 or 10, characterized in that the at least one through-flow channel (3) is designed such that its diameter varies in the flow direction (23).